A growing body of evidence suggests a causal relationship between the accumulation of classically activated macrophages in adipose tissue and development of insulin resistance in obesity. The molecular mechanisms regulating adipose tissue macrophage (ATM) activation state are poorly understood. Recent studies of peroxisome proliferator activated receptor-gamma (PPAR3) knockout mice indicate its importance for development of the 'alternative activation'state in ATMs. Alternatively activated macrophages exhibit low inflammatory potential, upregulate genes involved in fatty acid catabolism (2-oxidation) and function primarily in tissue repair. During progression to the obese state, however, there is a phenotypic switch from alternative activation to classical activation, in which ATMs produce abundant inflammatory mediators linked to insulin resistance. The mechanisms underlying PPAR3-dependent alternative activation and the phenotypic switch in obesity are largely unknown. Several recent studies have reported that ATMs are filled with cytoplasmic triglyceride (TG). Given that TG stores are continuously metabolized to FFAs and since FFAs are endogenous ligands for PPAR3, we reasoned accumulation of adipose tissue TG might play an important role in PPAR3-dependent alternative activation in ATMs. To test this hypothesis, we developed a novel model system that allows us to induce dose-dependent uptake of adipose tissue TG in cultured macrophages. Our preliminary studies indicate that TG accumulation has dose-dependent and striking effects on inflammatory cytokine production. While modest TG accumulation inhibits classical macrophage activation, higher levels of TG loading triggers inflammatory cytokine production. This is the first evidence to our knowledge that adipose tissue TG accumulation may play an important role in regulating the activation state of ATMs. The broad objective of this grant proposal is to begin understanding how TG modulates cell signaling pathways and inflammatory gene expression in macrophages. Specifically, we will 1) test the hypothesis that modest triglyceride accumulation promotes alternative macrophage activation through PPAR3-dependent mechanisms, and 2) test the hypothesis that fatty acid overload promotes classical macrophage activation. Our studies will provide novel insights into this poorly understood, but potentially very important, aspect of macrophage-adipocyte interactions in obesity. This work is significant to understanding the relationship between obesity, inflammation, and the innate immune system, particularly in regards to the role that macrophages play. PUBLIC HEALTH RELEVANCE A growing body of evidence suggests a causal relationship between macrophage accumulation in adipose tissue and insulin resistance in the obese state. We have developed a novel model system that will allow us to study how adipose tissue triglyceride accumulation, as occurs in vivo, modulates inflammatory gene expression in macrophages. Our studies are the first to explore the molecular mechanisms underlying this poorly understood, but potentially very important, niche of adipose tissue macrophage biology.